Banana is one of the world's most important fruit crops with a world production of approximately 98 million tons annually (FAO, 2001). However, as with many monocultures, a range of fungal, viral, bacterial and nematode diseases affects banana, which cause severe economical losses every year.
Fusarium wilt is one of the most destructive and notorious diseases of banana. It is also known as Panama disease, in recognition of the extensive damage it caused in export plantations in this Central American country. By 1960, Fusarium wilt had destroyed an estimated 40,000 ha of ‘Gros Michel’ (AAA), causing the export industry to convert to cultivars in the Cavendish subgroup (AAA) (Ploetz and Pegg, 2000). Fusarium wilt is caused by the soilborne hyphomycete, Fusarium oxysporum Schlect. f sp. cubense. It is one of more than 120 formae speciales (special forms) of F. oxysporum that cause vascular wilts of flowering plants. This pathogen affects species of Musa and Heliconia, and strains have been classified into four physiological races based on pathogenicity to host cultivars in the field (race 1, ‘Gros Michel’; race 2, ‘Bluggoe’; race 3, Heliconia spp.; and race 4, Cavendish cultivars and all cultivars susceptible to race 1 and 2). Until recently, race 4 had only been recorded to cause serious losses in the subtropical regions of Australia, South Africa, the Canary Islands, and Taiwan. If this race were to become established in the Americas, the world export industries would be severely affected, as there is no widely accepted replacement for Cavendish cultivars (Bentley et al., 1998).
In general, effective chemical control measures do not exist. In work conducted in South Africa, methyl bromide significantly reduced disease incidence, but was effective for only three years due to recolonisation of the fumigated areas by the pathogen. Studies on the biological and cultural control of this disease have begun only recently. Arbuscular mycorrhizal fungi have been shown to reduce disease severity in short-term green house studies, but results from long term field studies have not been reported (Ortiz et al., 1995). Tissue-culture plantlets are free of pathogens and should be used to establish new plantings whenever possible. However the expense of plantlets may make their use in subsistence agriculture impractical. Genetic resistance offers the greatest opportunity for managing this disease in infested soils (Ortiz et al., 1995).
Plants recognise and resist many invading pathogens by inducing a rapid defense response, termed the hypersensitive response (HR). The HR results in localised cell and tissue death at the site of infection, which constrains further spread of the infection. This local response often triggers non-specific resistance throughout the plant, a phenomenon known as systemic acquired resistance (SAR). Once triggered, SAR provides resistance to a wide range of pathogens for days. The HR and SAR depend on interaction between a dominant or semidominant resistance gene (R) product in the plant and a corresponding dominant phytopathogen avirulence gene (Avr) product (Baker et al., 1997). A loss or alteration to either the plant R gene or the pathogen Avr gene leads to disease (compatibility) (Hammond-Kosack and Jones, 1997).
The R proteins provide resistance to pathogens as diverse as fungi, bacteria, viruses, nematodes and insects. Eight classes of R genes have been defined according to the structural characteristics of their predicted protein: (1) cytoplasmic toxin reductase enzymes; (2) intracellular protein kinases; (3) receptor kinase-like protein with two tandem protein kinase domain; (4) receptor-like protein kinases with an extracellular leucine-rich repeat (LRR) domain; (5) intracellular LRR proteins with a nucleotide binding site (NBS) and leucine zipper (LZ) motif; (6) intracellular NBS-LRR proteins with a region with similarity to the Toll and interleukin-1 receptor (TIR) proteins; (7) LRR proteins that encode membrane-bound extracellular proteins; and (8) LZ proteins that encode membrane-bound intracellular proteins (FIG. 1). With a few exceptions, all R genes have been cloned by a map-based cloning approach.
The NBS-LRR class is by far the largest group of resistance proteins with more than 30 cloned genes to date. Two subgroups within the NBS-LRR class have been recognised by the presence or absence of ah amino N-terminal region (TIR domain) with amino acid sequence similarity to the cytoplasmic signalling domains of the Toll and interleukin-1 receptors (Meyer et al., 1999; Pan et al., 2000).
The N-terminal of some NBS-LRR proteins is similar to the cytoplasmic effector domain of the Drosophila melanogaster and human TOLL and interleukin-1 receptors (the TIR domain)(Hammond-kosack and Jones, 1997). Other NIBS-LRR proteins have different N-terminal domains, which often contain putative leucine-zipper (LZ) motifs. Mutational analysis in Arabidopsis revealed that TIR-NBS-LRR and LZ-NBS-LRR proteins employ different signalling pathways. Proteins in the TIR effector domain signal via a pathway that includes EDS1, a predicted lipase, whereas most LZ-NBS-LRR proteins examined employ the membrane-associated NDR1 protein (Aarts et al., 1998). There is no apparent correlation between pathogen type and the NBS-LRR subclass used by plants to detect these pathogens (Ellis and Jones 1998). All this evidence is consistent with the hypothesis of Aarts et al., (1998), who suggested that there may be two downstream pathways triggered by R genes, with the structure of the R protein determining which downstream factors are required. Other recent results have shown that the situation may not be this simple. Two R genes from Arabidopsis, RPP8 and RPP13 (both LZ-NBS-LRR proteins), require neither EDS1 nor NDR1, suggesting that there is at least a third pathway for the transduction of R-gene signals (Glazebrook, 2001). Although many studies on different R genes have suggested that the R-protein LRR domain makes the major contribution to the unique recognition capacity of individual R genes, recent analyses of the L allelic series has shown that the TIR domain can also contribute to this capacity. Thus, it is possible that the LRR are necessary but not sufficient for the specific recognition of Avr proteins and that LRR and amino-terminal domains have co-evolved to function in a coordinate manner. (Zachary, 2001).
The central NBS domain comprises three motifs predicted to bind ATP or GTP, and several conserved motifs whose functions are not known (Hammond-Kosack and Jones, 1997). This region has homology to two activators of apoptosis in animal cells: APAF-1 and CED. By analogy to these well-characterised regulators of programmed cell death, the corresponding domain in NBS-LRR proteins might operate as an intramolecular signal transducer (Van der Biezen and Jones, 1998; Aravind et al., 1999). Domain swaps involving several flax L alleles reveal a requirement for intramolecular interactions and, thus, NBS-LRR proteins might serve as adaptor molecules that link recognition and signal delivery. For example, Avr signals perceived by the LRR might initiate nucleotide hydrolysis at the NBS domain. This might provide the energy necessary for a confrontational change in the NBS-LRR protein, exposing its N-terminal effector portion, to trigger a defense response (Van der Biezen and Jones, 1998).
LRR domain is thought to be involved in ligand-binding and pathogen recognition. LRR contain leucines or other hydrophobic residues at regular intervals and can also contain regularly spaced prolines and asparagines (Bent, 1996). Comparative analyses of the LRR domain show hypervariability, suggesting diversification due to selection pressures. This indicates that recognition specificity resides in this part of the LRR. By analyses of in vivo and in vitro generated recombinants between different flax L alleles, Ellis et al. (1997) confirmed experimentally that the LRR constitute the principal determinant of specificity for Avr products. Differential specificities of R proteins are often associated with duplications, deletions and sequence exchanges within the regions that encode the LRR. Recently, the LRR-like domain of the rice resistance protein Pita was shown to be required for interaction with Avr-Pita in the yeast two-hybrid system. Furthermore, mutation in either Avr-Pita Pita that abolished resistance also abolished the interaction in vitro. This is the first demonstrated interaction between an LRR-containing R protein and its cognate Avr protein (Jia et al., 2000).
Some of the resistance genes isolated to date have been transferred to susceptible cultivars of the same species or different species with successful results. For example, the N gene for resistance to Tobacco mosaic virus (TMV) has been transferred to tomato and gives resistance in this species to TMV (Whitham et al., 1996). The Bs2 gene, which encodes Xanthomonas resistance in pepper, has been cloned and transferred to tomato, a crop species in which the number of useful resistance genes to this pathogen is limited (Tai et al., 1999). However, the RPS2 gene from Arabidopsis is non-functional in transgenic tomato and this phenomenon has been referred to as ‘restricted taxonomic functionality’ (Tai et al., 1999). These data suggest that there may be difficulties in wide, cross-species resistance-gene transfer, in certain instances, due to R gene specificity Ellis et al., 2000).
The ability to isolate and transfer R genes eliminates the issue of retention of unwanted and genetically linked germoplasm, an important problem associated with classical breeding. Although disease-resistance transgenic plants are no yet available commercially, future product development seems likely as our current level of understanding of pathogenesis and plant defense improves (Stuiver and Custers 2002).
Despite the progress in R gene biology, however, no resistance genes have been isolated to date, which can confer resistance to destructive banana diseases in susceptible cultivars.
In work leading up to the present invention, four genotypes of banana were investigated to identify candidate R genes that would confer resistance to race 4 of Fusarium oxysporum fsp cubense. These genotypes were as follows: Cavendish, which is resistant to race 1 but susceptible to race 4; Calcutta 4, which is resistant to race 1 and race 4; three progeny of Musa acuminata spp malaccensis, which are susceptible to race 4; and three progeny of Musa acuminata spp malaccensis, which are resistant to race 4. Five families of R genes were identified from this investigation, all of which were present in the genomes of each of the genotypes but which had slightly different sequences. Surprisingly, two of these families (RGA2 and RGA5) were found to share some sequence similarity with the I2 R gene, which confers resistance to Fusarium wilt in tomatoes. In addition RGA2 was shown to be transcribed in the three resistant Musa acuminata spp malaccensis progeny but not in the three susceptible progeny. These discoveries have been reduced to practice in compositions and methods for modulating disease resistance, especially fungal resistance, in plants including banana and in plants and plant parts, especially genetically modified plants, plant cells, tissues and seeds, having modified disease resistance, as described hereafter.